The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

Abstract

Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a ∼600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5′-aminolevulinic acid dehydratase (ALAD, the second step). Similarly, Escherichia coli hemH (FC) deficient strains transformed with human and Wolbachia FC homologues showed significantly different sensitivities to NMMP. This approach enables functional complementation in E. coli heme deficient mutants as an alternative E. coli-based method for drug screening. Our studies indicate that the heme biosynthetic genes in the Wolbachia of B. malayi (wBm) might be essential for the filarial host survival. In addition, the results suggest they are likely candidate drug targets based upon significant differences in phylogenetic distance, biochemical properties and sensitivities to heme biosynthesis inhibitors, as compared to their human homologues. Human filarial nematodes are causative agents of elephantiasis and African river blindness, which are among the most debilitating tropical diseases. Currently used drugs mainly affect microfilariae (mf) and have less effect on adult filarial nematodes, which can live in the human host for more than a decade. Filariasis drug control strategy relies on recurrent mass drug administration for many years. Development of novel drugs is also urgently needed due to the threat of drug resistance occurrence. Most filarial worms harbor an obligate endosymbiotic bacterium, Wolbachia, whose presence has been identified as a potential drug target. Comparative genomics had suggested Wolbachia heme biosynthesis as a potential drug target, and we present an analysis of selected enzymes alongside their human homologues from several different aspects—gene phylogenetic analyses, in vitro enzyme kinetic and inhibition assays and heme-deficient E. coli complementation assays. We also conducted ex vivo Brugia malayi viability assays using heme pathway inhibitors. These experiments demonstrate that heme biosynthesis could be critical for filarial worm survival and thus is a potential anti-filarial drug target set.